It is unclear if epithelial cells can defend against invading bacteria. During years 01-04, calprotectin (S100A8/S100A9; MRPB-MRP14) expression was shown to reduce invasion into epithelial cells by L. monocytogenes, S. typhimurium, and P. gingivalis. Resistance was associated with: i) increased a3-integrin expression; ii) altered actin organization, and iii) intracellular antimicrobial activity. Calprotectin is posited to be a multifunctional protein complex that reduces bacterial invasion by several mechanisms. Aim 1 will express defined mutants of calprotectin complex in KB cells and analyze for altered function. Site-directed mutational analysis and deletion analysis will identify structural features of calprotectin required for inhibition of invasion. Aim 2: Regulate MRP8 (S100A8) and MRP14 (S100A9) expression in KB cells using an inducible mammalian expression system. Experimental invasion will occur: 1) before cytoplasmic expression of calprotectin, 2) during half-maximal expression, and 3) when calprotectin is maximal. Calprotectin will be localized from the site of synthesis to other cell compartments over time in the presence and absence of invasion. These studies will show the staged movement of calprotectin within the cell, the relationship to the invasion of different species, and whether bacteria actually invade before inhibition by calprotectin. Aim 3: Target calprotectin to selected subcellular locations to enhance or reduce its anti-invasion action. Calprotectin will be over-expressed in selected subcellular and extracellular compartments and under-expressed in the cytoplasm. Cells over-expressing membrane and secreted calprotectin (under-expressed in the cytoplasm) will be compared to normal calprotectin-expressing cells for anti-invasion and anti-microbial activity. If cytoplasmic interactions were crucial, membrane or secreted calprotectin would be ineffective in inhibiting invasion. AIM 4: Compare human gingival epithelial cells and KB cells for the effect of calprotectin on bacterial invasion. Human gingival epithelial cells will be incubated with PMA to up-regulate calprotectin, or retinoic acid, DMSO or vitamin D3 to down-regulate expression. After dose-response optimization, and comparison to KB cells to identify non-specific changes in invasion phenotype, calprotectin will be localized. Aim 4 will bridge molecular mechanisms of innate resistance to bacterial invasion in stable transfectants to normal epithelial cells in vitro. These Aims will show the multifunctional potential for calprotectin to make mucosal epithelial cells resistant to bacterial invasion in vitro, suggesting new mechanisms of innate immunity in vivo and functional targets for new anti-bacterial therapies.